1. Field of the Invention
This invention is related to the field of probe-based detection, analysis and quantitation of nucleic acids which are electrostatically immobilized to matrices. The methods, kits and compositions of this invention are particularly well suited for the analysis, and particularly single point mutation analysis, in a particle assay, in an array assay, in a nuclease digestion/protection assay, in a line assay and/or in a self-indicating assay format.
2. Description of the Related Art
Nucleic acid hybridization is a fundamental process in molecular biology. Probe-based assays are useful in the detection, quantitation and analysis of nucleic acids. Nucleic acid probes have long been used to analyze samples for the presence of nucleic acid from bacteria, eucarya, fungi, virus or other organisms and are also useful in examining genetically-based disease states or clinical conditions of interest in single cells as well as in tissues.
Sample prep methods which describe the repetitive capture and release of target sequences to and from supports (e.g. magnetic beads) as a means to remove non-target polynucleotides, debris and impurities which tend to introduce background in a hybridization assay are known in the art (See: Collins et al., U.S. Pat. No. 5,750,338). Generally, the sample prep methods of Collins et al. can be used in most embodiments of traditional hybridization assays provided however that the target nucleic acid is first immobilized to a support and thereafter released from the support such that, when released, it is substantially free of sample impurities, debris, and extraneous polynucleotides. The Collins et al. invention, however, requires that the probe or probes must be associated with or capable of associating with the support under binding conditions to thereby immobilize the nucleic acid of interest to the support (See: Collins et al. at col. 4, line 55 to col. 5, line 13).
A probe based sample prep method for removing contaminants prior to PCR reaction has been described by Goldin et al. (See: U.S. Pat. No. 5,200,314). This process requires an analyte-capture probe having both an analyte binding region and a first specific binding partner. Like the Collins et al. invention, the Goldin et al. invention requires that the analyte-capture probe interact with the support as the specific means through which the target sequence becomes immobilized.
Polycationic solid supports have been used for the analysis and purification of nucleic acids, including the purification of polynucleotides from solutions containing contaminants See: Arnold et al.; U.S. Pat. No. 5,599,667). Arnold et al. describe assays which use solid supports as a means to separate polynucleotides, and hybrids thereof formed with a nucleotide probe, from unhybridized probe (See: Abstract to U.S. Pat. No. 5,599,667). The invention is premised upon xe2x80x9c. . . the discovery that polycationic solid supports can be used to selectively adsorb nucleotide multimers according to their size (emphasis added), larger multimers being more tightly bound to the support than smaller ones.xe2x80x9d (See: Col. 4, lines 39-44). The methods can also be used to separate the nucleotide multimers from non-nucleotidic material (See: Col. 5, lines 25-28).
A substantial limitation of the Arnold et al. invention is the interplay which exists between the composition of the cationic solid support and the formulation of contacting solutions as well as the interplay between two or more of the contacting solutions (See: Col. 7, line 24 to Col. 8, line 32) which are required to discriminate between nucleotide multimers (See: Col. 8, lines 39-41). An example of a laborious protocol for arriving at a proper cation density for a solid support can be found at col. 9, lines 36-52 and the method for determining the buffer concentration suitable for separating polynucleotides and nucleotide probes can be found at col. 9, lines 53-63. Similarly, the separation solution must be carefully designed (See: Col. 10. lines 9-12), presumably using the laborious method of trial and error as described for determining the cation density of the solid support. This requirement for substantial optimization of assay conditions within a very narrow operating range results because electrostatic immobilization of nucleic acid is a relatively non-specific process and therefore it is difficult to electrostatically immobilize a negatively charged target nucleic acid to a cationic surface without the positively charged matrix also exhibiting a strong affinity for the negatively charged nucleic acid probe. Since the separation of nucleotide multimers (nucleotide probe/target hybrids from excess nucleotide probe) occurs within a narrow range of conditions, which may not necessarily be optimal for the discrimination of hybridization, the hybrids still immobilized according to the Arnold et al. invention may not be truly indicative of the presence of a target sequence. Consequently, the applicability of the assays of Arnold et al. are of limited practical utility.
An invention related to achieving nucleic acid has recently been described (See: Gerdes et al.; WO98/46797). Gerdes et al. use highly electropositive solid phase materials to capture nucleic acids (See. p. 5, line 24 to p. 6, line 14) for repetitive analyses. However, a substantial limitation of the Gerdes et al. invention is that the nucleic acid must be irreversibly bound to the highly electropositive solid phase material.
Methods for the high throughput screening for sequences or genetic alterations in nucleic acid have been described (See: Shuber, A. P.; U.S. Pat. No. 5,834,181). Shuber describes the analysis of arrays of immobilized nucleic acids, and suggests immobilization of the nucleic acid to nitocellulose or a charged nylon membrane (See: col. 6, lines 41-64). Suggested purine and pyrimidine containing polymers which may be used for analyzing immobilized nucleic acid include peptide nucleic acid (See: col. 5, lines 15-20), but the polymers must necessarily be tagged or labeled since the detection methods rely on a tag or label being incorporated into the polymer (See: col. 8, line 58 to col. 9, line 3). The assays of Shuber require a perfect complement between probe and target sequence (See: col. 8, lines 52-57). In order to achieve proper discrimination, a laborious empirical process of trial and error is described for assay optimization (See: col. 7, line 16 to col. 8). Conditions which require optimization of specific and non-specific hybridization include the concentration of polymer, the temperature of hybridization, the salt concentration, and the presence or absence of unrelated nucleic acid (See: col. 8, lines 15-18).
Shuber does not expressly suggest performing a probe-based hybridization assay on an electrostatically immobilized nucleic acid and specifically does not describe or teach the analysis of electrostatically immobilized nucleic acid using a non-nucleotide probe such as a peptide nucleic acid. Furthermore, Shuber does not suggest, disclose or teach any advantages, such the ability to work within a broad range of assay conditions, of performing a peptide nucleic acid-based analysis of nucleic acid electrostatically immobilized to a matrix.
Pluskal et al. describe a comparison of DNA and peptide nucleic acid (PNA) probe-based analysis of nucleic acid which has been irreversibly crosslinked to charged nylon membrane (See: Pluskal et al., American Society for Biochemistry, 85th Annual Meeting, Washington, D.C., May 1994). Pluskal et al. teach that while PNA probes can be used to detect the irreversibly immobilized nucleic acid under standard hybridization conditions, PNA works very well under highly stringent hybridization and washing conditions (See: The Section Entitled xe2x80x9cDiscussionxe2x80x9d). Pluskal et al. also teach the use of 1% BSA as a blocking agent to reduce non-specific binding of the probe to the membrane (See: Section Entitled xe2x80x9cDiscussionxe2x80x9d). Because the nucleic acid of Pluskal et al. has been irreversibly crosslinked to the nylon membrane, highly stringent hybridization and washing conditions can be applied to the membrane without reducing the amount of target nucleic acid present on the support and available for analysis. Pluskal et al. therefore demonstrate a rationale for irreversibly linking the nucleic acid to be analyzed to the support and using a blocking agent when performing a PNA probe-based analysis using a charged nylon membrane.
Methods for the protection of nucleic acid sequences from nuclease degradation/digestion by hybridizing a nucleic acid analog thereto have been described (See: Stanley et al.; U.S. Pat. No. 5,861,250). The methods and compositions described in Stanley et al. are particularly well suited for xe2x80x9ccleaning upxe2x80x9d a nucleic acid sample by degrading all nucleic acid present except the target sequence, . . . xe2x80x9d (See: Stanley et al. at col. 7, lines 14-18). Stanley et al. describe several means for separating hybridized nucleic acid analog from non-hybridized nucleic acid analog, including ion exchange chromatography (See: Col. 6, lines 62-64), but they do not describe the simple electrostatic immobilization of the target sequence or nucleic acid analog/target sequence complex to a matrix as means to separate the hybridized nucleic acid analog from non-hybridized nucleic acid analog or otherwise separate the nucleic acid analog/target sequence complex from the other components of a sample.
Methods and apparatus for the electroactive transport and fixation of nucleic acids for analysis have been described (See: Heller et al., U.S. Pat. No. 5,849,486). However, this invention requires highly sophisticated instrumentation and devices to transport, fix and/or analyze a sample.
Though van den Engh does not discuss the detection of complex macromolecules such as nucleic acids, fluorescent reporter beads and methods for detecting the presence or determining the concentration of fluid bulk analytes such as pH, oxygen saturation and ion content are known in the art (See: van den Engh et al., U.S. Pat. No. 5,747,349). According to van den Engh, xe2x80x9cReporter beads are added to a fluid sample and the analyte concentration is determined by measuring fluorescence of individual beads, for example in a flow cytometerxe2x80x9d (See: Abstract of U.S. Pat. No. 5,747,349). The beads of van den Engh et al. comprise a substrate bead having a plurality of fluorescent reporter molecules immobilized thereon wherein the fluorescent reporter molecules comprise a fluorescent molecule whose fluorescent properties are a function of the concentration of the particular analyte whose presence or concentration is to be determined (See: U.S. Pat. No. 5,747,349 at col. 3, Ins. 29-46). Thus, the beads of van den Engh are inherently fluorescent and not the analytes or derivatives thereof.
Recently, compositions containing at least one bead conjugated to a solid support and further conjugated to at least one macromolecule have been described in the art (See: Lough et al., PCT/US97/20194). Claimed advantages of Lough et al. include increased surface area for the immobilization of biological particles or macromolecules as compared to flat surfaces as well as the ability to use one chemistry for the immobilization of the macromolecule to the bead and a different chemistry to attach the bead to the support. Lough et al. define macromolecules to include nucleic acids (See: p. 7, Ins. 10-17) and further define peptide nucleic acids (PNA) as being analogs of nucleic acids (See: p. 8, Ins. 4-9). The invention of Lough et al. is primarily directed to analysis of immobilized macromolecules. Curiously however, a probe-based assay is not described as a detection method but rather Lough et al. focus on direct analysis of the immobilized macromolecule be means such as MALDI-TOF mass spectrometry. Apart from apparently being considered by Lough et al. to be an analog of a nucleic acid, PNA is not otherwise mentioned in the disclosure and no examples are provided which demonstrate that PNA is suitable for the practice of the invention.
Despite its name, Peptide Nucleic Acid (PNA) is neither a peptide, a nucleic acid nor is it an acid. Peptide Nucleic Acid (PNA) is a non-naturally occurring polyamide which can hybridize to nucleic acid (DNA and RNA) with sequence specificity (See: U.S. Pat. Nos. 5,539,082, 5,527,675, 5,623,049, 5,714,331, 5,736,336, 5,773,571 or 5,786,461 as well as Egholm et al., Nature 365: 566-568 (1993)). Being a non-naturally occurring molecule, unmodified PNA is not known to be a substrate for the enzymes which are known to degrade peptides or nucleic acids. Therefore, PNA should be stable in biological samples, as well as have a long shelf-life. Unlike nucleic acid hybridization which is very dependent on ionic strength, the hybridization of a PNA with a nucleic acid is fairly independent of ionic strength and is favored at low ionic strength, conditions which strongly disfavor the hybridization of nucleic acid to nucleic acid (Egholm et al., Nature, at p. 567). The effect of ionic strength on the stability and conformation of PNA complexes has been extensively investigated (Tomac et al., J. Am. Chem. Soc. 118:55 44-5552 (1996)). Sequence discrimination is more efficient for PNA recognizing DNA than for DNA recognizing DNA (Egholm et al., Nature, at p. 566). However, the advantages in point mutation discrimination with PNA probes, as compared with DNA probes, in a hybridization assay, appears to be somewhat sequence dependent (Nielsen et al., Anti-Cancer Drug Design 8:53-65, (1993) and Weiler et al., Nucl. Acids Res. 25: 2792-2799 (1997)).
Because the nucleic acids of a complex sample, such as a cell lysate or PCR reaction mixture, can be concentrated and also partially purified by immobilization to supports, probe-based hybridization assays could be simplified if the presence of a target nucleic acid could be specifically detected while the target nucleic acid remained support bound; particularly if the conditions for the treatment, analysis and/or detection of the target sequence were operable within a broad range so that assay conditions did not require substantial and laborious optimization. The ability to perform such analyses using a flow cytometer, an array, a nuclease digestion/protection assay, a line assay, a self-indicating assay or in some combination of these assay formats would be particularly beneficial.
This invention pertains to methods, kits and compositions suitable for the detection, identification and/or quantitation of nucleic acids which are electrostatically immobilized to matrices using non-nucleotide probes which sequence specifically hybridize to one or more target sequences of the nucleic acid but do not otherwise substantially interact with the matrix. Once the nucleic acid is immobilized, the detectable non-nucleotide probe/target sequence complex, formed before or after the immobilization of the nucleic acid, can be detected, identified or quantitated under a wide range of assay conditions as a means to detect, identify or quantitate the target sequence in the sample. Because it is reversibly bound, the non-nudeotide probe/target sequence can optionally be removed from the matrix for detecting, identifying or quantitating the target sequence in the sample. Because the non-nucleotide probe/target sequence is protected against degradation, it is another advantage of this invention that the sample can be treated with enzymes which degrade sample components, either before or after the nucleic acid is bound to the matrix, in order to xe2x80x9cclean upxe2x80x9d the sample (e.g. a complex biological sample such as a cell lysate) and thereby improve the detection, identification or quantitation of the target sequence in the sample. Consequently, the methods, kits and compositions of this invention have substantial advantages over all previously known or described methods, kits or compositions because they facilitate the simple processing and/or analysis of samples, and particularly complex biological samples, under a wide range of assay conditions.
In one embodiment, this invention is related to a composition comprising a nucleic acid, having at least one target sequence, which is electrostatically bound to a matrix under suitable electrostatic binding conditions. The composition further comprises a detectable, but not necessarily labeled, non-nucleotide probe having a probing nucleobase sequence which is sequence specifically hybridized to at least a portion of the target sequence.
In another embodiment, this invention pertains to methods for the detection, identification or quantitation of a target sequence in a sample containing nucleic acid. One exemplary method comprises contacting a sample with a matrix and at least one non-nucleotide probe wherein the nucleic acid in the sample will electrostatically bind to the matrix under suitable electrostatic binding conditions. Additionally, the non-nucleotide probe will hybridize, under suitable hybridization conditions, to at least a portion of the target sequence, if present in the sample. The method further comprises detecting, identifying or quantitating the non-nucleotide probe/target sequence hybrid as a means to detect, identify or quantitate the target sequence in the sample.
In still another embodiment, this invention pertains to multiplex methods for the detection, identification or quantitation of two or more target sequences of one or more nucleic acid molecules which may be present in the same sample. One exemplary method comprises contacting a sample with a matrix and two or more independently detectable non-nucleotide probes wherein the nucleic acid present in the sample will electrostatically bind to the matrix under suitable electrostatic binding conditions. Additionally, the two or more independently detectable non-nucleotide probes will hybridize, under suitable hybridization conditions, to at least a portion of the target sequences with which each probe is designed to hybridize if present in the nucleic acid of the sample. Consequently, if a particular target sequence is electrostatically immobilized to the matrix, the independently detectable non-nucleotide probe designed to hybridized to that particular target sequence will become concentrated on the matrix and be available for detection. Therefore, the method further comprises detecting, identifying or quantitating each unique independently detectable non-nucleotide probe/target sequence hybrid which is electrostatically bound to said matrix as a means to detect, identify or quantitate each unique target sequence sought to be detected in the sample and in the same assay. Optionally, the unique independently detectable non-nucleotide probe/target sequence hybrid is released from the matrix by adjustment of conditions outside the range required for electrostatic binding and thereby facilitates detection of the unbound non-nucleotide probe/target sequence hybrid, or just the detectable probe, as the means to detect, identify or quantitate the target sequence in the sample.
In still a further embodiment, this invention takes advantage of the stability of nucleic acid analog/nucleic acid complexes (See: Stanley et al.; U.S. Pat. No. 5,861,250) to thereby further improve assay performance and/or otherwise decrease the labor or complexity of sample preparation. One exemplary method comprises contacting the sample with at least one non-nucleotide probe wherein the non-nucleotide probe will hybridize, under suitable hybridization conditions, to at least a portion of the target sequence if present in the sample. The sample is also contacted with a matrix wherein the nucleic acid molecule will electrostatically bind to a matrix under suitable electrostatic binding conditions. Either before or after immobilization to the matrix, the sample containing the non-nucleotide probe/target sequence complex is contacted with one or more enzymes capable degrading sample contaminants possibly including the nucleic acid molecule but not the non-nucleotide probe/target sequence complex. The method further comprises detecting, identifying or quantitating the non-nucleotide probe/target sequence hybrid as a means to detect, identify or quantitate the target sequence in the sample. Optionally, the detectable non-nucleotide probe/target sequence hybrid is released from the matrix by adjustment of conditions outside the range required for electrostatic binding and thereby facilitates detection of the unbound non-nucleotide probe/target sequence hybrid, or just the detectable probe, as the means to detect, identify or quantitate the target sequence in the sample.
In yet another embodiment, this invention relates to a method for the detection, identification or quantitation of a target sequence of a nucleic acid molecule electrostatically immobilized at a location on an array wherein the array comprises nucleic acid molecules electrostatically bound at unique locations. One exemplary method comprises contacting the array with at least one non-nucleotide probe, wherein the non-nucleotide probe will hybridize, under suitable hybridization conditions, to at least a portion of the target sequence if present on the array. The non-nucleotide probe/target sequence complex electrostatically bound at a location on said array is then detected, identified or quantitated as the means to determine the presence, absence or amount of target sequence present at said array location. It is an advantage of the invention that one or more enzymes capable of degrading sample contaminants including the nucleic acid target molecule but not the non-nucleotide probe/target sequence complex, can also be added before analysis of the array to thereby improve the performance of the array assay by degrading sample contaminants which might otherwise lead to false positive results. Optionally, the detectable non-nucleotide probe/target sequence hybrids can be released from the matrix by adjustment of conditions outside the range required for electrostatic binding and thereby facilitates detection of the unbound non-nucleotide probe/target sequence hybrid, or just the detectable probe, as the means to detect, identify or quantitate target sequence in the sample. If the non-nucleotide probes are independently detectable, the analysis of the matrix can proceed in a multiplex format.
In yet a further embodiment, this invention is directed to a method for the detection, identification or quantitation of a target sequence of a nucleic acid molecule which may be present in any of two or more samples of interest. The method comprises mixing each of the two or more samples of interest with at least one non-nucleotide probe, under suitable hybridization conditions. Next a matrix is contacted, under suitable electrostatic binding conditions, with at least a portion of each of the two or more samples to thereby electrostatically immobilize the nucleic acid components of each sample to the matrix, each at a unique location, and thereby create a matrix array of samples. The non-nucleotide probe/target sequence complex electrostatically bound at a location on said array is then detected, identified or quantitated as the means to determine the presence, absence or amount of target sequence present at said array location. It is an advantage of the invention that one or more enzymes capable of degrading sample contaminants including the nucleic acid target molecule but not the non-nucleotide probe/target sequence complex, can also be added before analysis of the array to thereby improve the performance of the array assay by degrading sample contaminants which might otherwise lead to false positive results. Optionally, the detectable non-nucleotide probe/target sequence hybrids can be released from the matrix by adjustment of conditions outside the range required for electrostatic binding and thereby facilitates detection of the unbound non-nucleotide probe/target sequence hybrid, or just the detectable probe, as the means to detect, identify or quantitate target sequence in the sample. If the non-nucleotide probes are independently detectable, the analysis of the matrix can proceed in a multiplex format.
In yet another embodiment, this invention is directed to kits suitable for performing an assay which detects the presence, absence or number of target sequences present in a sample. The kits of this invention comprise a matrix and one or more non-nucleotide probes and optionally one or more other reagents or compositions which are selected to perform an assay of this invention or otherwise simplify the performance of an assay used to detect, identify or quantitate a target sequence in a sample.
The compositions, methods and kits of this invention are particularly useful for the detection, identification and/or enumeration of bacteria and eucarya (e.g. pathogens) in food, beverages, water, pharmaceutical products, personal care products, dairy products or environmental samples. The analysis of preferred non-limiting beverages include soda, bottled water, fruit juice, beer, wine or liquor products. Suitable compositions, methods and kits will be particularly useful for the analysis of raw materials, equipment, products or processes used to manufacture or store food, beverages, water, pharmaceutical products, personal care products dairy products or environmental samples.
Additionally, the compositions, methods and kits of this invention are particularly useful for the detection of bacteria and eucarya (e.g. pathogens) in clinical samples and clinical environments. Suitable compositions, methods and kits will be particularly useful for the analysis of clinical specimens, equipment, fixtures or products used to treat humans or animals.